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Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation Via Decrease in Osteoblasts

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Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation Via Decrease in Osteoblasts International Journal of Molecular Sciences Article Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts Kazunori Hamamura 1,* , Kosuke Hamajima 1,2, Shoyoku Yo 1,2, Yoshitaka Mishima 1,2, Koichi Furukawa 3 , Makoto Uchikawa 4, Yuji Kondo 5, Hironori Mori 1,2, Hisataka Kondo 1, Kenjiro Tanaka 1, Ken Miyazawa 2, Shigemi Goto 2 and Akifumi Togari 1 1 Department of Pharmacology, School of Dentistry, Aichi Gakuin University, Nagoya 464-8650, Japan; [email protected] (K.H.); [email protected] (S.Y.); [email protected] (Y.M.); [email protected] (H.M.); [email protected] (H.K.); [email protected] (K.T.); [email protected] (A.T.) 2 Department of Orthodontics, School of Dentistry, Aichi Gakuin University, Nagoya 464-8650, Japan; [email protected] (K.M.); [email protected] (S.G.) 3 Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Aichi 487-8501, Japan; [email protected] 4 Japanese Red Cross Tokyo Blood Center, Tokyo 162-8639, Japan; [email protected] 5 Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 464-8650, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-52-757-6743; Fax: +81-52-752-5988 Received: 27 August 2019; Accepted: 17 September 2019; Published: 18 September 2019 Abstract: Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation. Keywords: glycosphingolipids; globoside (Gb4); osteoblasts; bone metabolism 1. Introduction Glycosphingolipids are considered to play an important role in the development and maintenance of various organs and tissues [1–3]. These have also been shown to be critical for protection against Int. J. Mol. Sci. 2019, 20, 4619; doi:10.3390/ijms20184619 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 4619 2 of 13 Int. J. Mol. Sci. 2019, 20, x 2 of 13 inflammationGlycosphingolipids and degeneration are considered [4]. Glycosphingolipids to play an important are divided role in into the three development main series, and i.e., ganglioside-series,maintenance of globo-series, various organs and and lacto tissues/neolacto-series. [1–3]. These Ofhave these, also gangliosides been shown haveto be beencritical reported for to beprotection involved inagainst bone inflammation metabolism and [5– 9degeneration]. For instance, [4]. Glycosphingolipids it was reported thatare divided a-series into ganglioside three GD1amain and GM1series, promote i.e., ganglioside osteoblastic‐series, di ffgloboerentiation‐series, and of bone lacto/neolacto marrow‐ mesenchymalseries. Of these, stem gangliosides cells (MSCs) and tendonhave been stem reported cells [to5– be8]. involved Recently, in bone we demonstrated metabolism [5–9]. that For a instance, lack of GD3it was synthase, reported that which a‐ is responsibleseries ganglioside for the generation GD1a and of GM1 all b-seriespromote osteoblastic gangliosides, differentiation in mice results of bone in marrow the attenuation mesenchymal of bone stem cells (MSCs) and tendon stem cells [5–8]. Recently, we demonstrated that a lack of GD3 synthase, loss with aging [9]. However, there have been no reports on the presence or absence of expression of which is responsible for the generation of all b‐series gangliosides, in mice results in the attenuation globo-series glycosphingolipids in osteoblasts and osteoclasts, and the roles of these glycosphingolipids of bone loss with aging [9]. However, there have been no reports on the presence or absence of in boneexpression metabolism. of globo‐series glycosphingolipids in osteoblasts and osteoclasts, and the roles of these Globo-seriesglycosphingolipids glycosphingolipids in bone metabolism. have a common core structure, Galα1,4-Galβ1,4-Glc-ceramide (globotriaosylceramide;Globo‐series glycosphingolipids Gb3), which is have synthesized a common by coreα 1,4-galactosyltransferasestructure, Galα1,4‐Galβ1,4‐Glc (Gb3‐ceramide synthase A4galt(globotriaosylceramide;) from lactosylceramide Gb3), (LacCer) which (Figure is synthesized1). Globo-series by α1,4‐ glycosphingolipidsgalactosyltransferase such(Gb3 assynthase Gb3, Gb4 (globoside),A4galt) andfrom Gb5 lactosylceramide (galactosyl (LacCer) globoside) (Figure have 1). not Globo only‐series been glycosphingolipids used as markers such for as stem Gb3, cells Gb4 and tumors(globoside), but are also and considered Gb5 (galactosyl to regulate globoside) immune have system not only and been maintain used as stemness markers [ 10for– 13stem]. Gb3 cells has and been characterizedtumors but as theare Palsok antigen considered on red to regulate blood cells immune [14] and system is considered and maintain to bestemness a tumor [10–13]. antigen Gb3 of has Burkitt been characterized as the Pk antigen on red blood cells [14] and is considered to be a tumor antigen lymphoma [10]. This glycosphingolipid has also been recognized as a receptor for verotoxins secreted of Burkitt lymphoma [10]. This glycosphingolipid has also been recognized as a receptor for by pathogenic Escherichia coli O157 [15]. Gb4 is a major component among glycosphingolipids in human verotoxins secreted by pathogenic Escherichia coli O157 [15]. Gb4 is a major component among erythrocytesglycosphingolipids [16] and is anin human essential erythrocytes structure [16] for bloodand is an group essential P antigen structure [17 for]. Gb4 blood was group also P reportedantigen to be an[17]. endogenous Gb4 was ligand also forreported Toll-like to be receptor an endogenous 4-myeloid ligand differentiation for Toll‐like factor receptor 2 (TLR4-MD-2) 4‐myeloid and attenuatesdifferentiation lipopolysaccharide factor 2 (TLR4 (LPS)‐MD toxicity‐2) and [ 13attenuates]. Gb5, whichlipopolysaccharide is also named (LPS) stage-specific toxicity [13]. embryonic Gb5, antigen-3which (SSEA-3), is also isnamed expressed stage‐specific at an early embryonic developmental antigen‐3 stage(SSEA and‐3), isis usedexpressed as a marker at an early for stem cells [11developmental]. stage and is used as a marker for stem cells [11]. LacCer Gb3 Gb3 synthase cer cer Gb4 synthase Gb4 (globoside) Gb5 Gb5 (SSEA-3) synthase cer cer cer : ceramide : Glucose : Galactose : N-acetylgalactosamine Figure 1. Synthetic pathway of globo-series glycosphingolipids. Figure 1. Synthetic pathway of globo‐series glycosphingolipids. In theIn present the present study, study, we we used used flow flow cytometry cytometry to to examine examine the the expression expression levels levels of ofglobo globo-series‐series glycosphingolipidsglycosphingolipids (Gb3, (Gb3, Gb4, Gb4, and and Gb5) Gb5) in in MC3T3 MC3T3 E1E1 mousemouse osteoblast osteoblast-like‐like cells, cells, SaM SaM-1‐1 human human osteoblastosteoblast cells, cells, RAW264.7 RAW264.7 mouse mouse pre-osteoclasts, pre‐osteoclasts, and and primary primary cultured cultured pre pre-osteoclasts‐osteoclasts derived derived from from mousemouse bone bone marrow marrow cells. cells. To evaluateTo evaluate the the involvement involvement ofof globo-seriesglobo‐series glycosphingolipids glycosphingolipids in bone in bone metabolismmetabolismin vivo in, vivo, femoral femoral cancellous cancellous bone bone mass mass in wild in wild type type (WT) (WT) and and Gb3 Gb3 synthase-knockout synthase‐knockout (Gb3S KO) mice(Gb3S were KO) analyzed mice were using analyzed three-dimensional using three‐dimensional micro-computed micro‐ tomographycomputed tomography (3D-µCT). (3D Furthermore,‐μCT). we conductedFurthermore, calcein we double conducted labeling calcein to examine double thelabeling roles ofto globo-series examine the glycosphingolipids roles of globo‐series in bone glycosphingolipids in bone formation. We employed bone histomorphometric analyses using formation. We employed bone histomorphometric analyses using hematoxylin and eosin (HE) and hematoxylin and eosin (HE)
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